This invention relates to nucleic acid and amino acid sequences of a CAF 1-related protein and to the use of these sequences in the diagnosis, treatment, and prevention of disorders associated with cell proliferation and inflammation.
Differential control of gene expression is essential to the growth and development of all multicellular organisms. Although gene expression can be controlled at many steps along the path from DNA to protein, the major control point for most genes is at the initiation of transcription. This critical step is regulated both positively and negatively by a combination of general and tissue specific transcription factors, the majority of which function to stimulate transcription of one or more target genes.
Many transcription factors are modular proteins that contain separable DNA binding and transcriptional activation (or repression) domains. The DNA binding domain interacts with specific DNA sequences (control elements) near the promoter region of the gene; this interaction brings the activation(or repression) domain into a position where it can interact with other proteins to stimulate (or repress) transcription. Many transcription factors require dimerization or multimerization to be fully functional. For example, members of the helix-loop-helix family of transcription factors function as homo-/or hetero-dimers. The monomeric forms of these factors lack DNA binding activity. (Stryer, L. (1995) Biochemistry, 4th ed., pp 998-999.)
CCR4 is a general transcription factor in yeast that appears to be a component of a multisubunit complex. CCR4 stimulates the expression of numerous genes involved in non-fermentative growth. In particular, CCR4 is required for expression of the glucose-repressible alcohol dehydrogenase II gene (ADH2). Although CCR4 does not appear to bind DNA directly, when fused to the DNA binding domain of LexA, CCR4 can function as a glucose responsive transcriptional activator. CCR4 physically interacts with several other protein factors. Two of these CCR4 associated factors, CAF1 and CAF2, bind to a leucine rich repeat motif in the middle of the CCR4 protein. (Denis, C. L. and Malvar, T. (1990) Genetics 124: 283-291; Malvar et al. (1992) Genetics 132 (4):951-962; Draper, M. P. et al. (1994) Mol. Cell. Biol. 14(7): 4522-4531; and Draper, M. P. et al. (1995) Mol. Cell. Biol. 15(7): 3487-3495.)
CAF1 is an evolutionarily conserved mouse protein, with homologs identified in human, S. cerevisiae, C. elegans, and A. thaliana. A yeast homolog of CAF1, POP2, was first identified by its effects on glucose regulated gene expression. Consistent with a proposed function as a transcription factor, both mouse CAF1 and yeast CAF1 can activate transcription of a LexA responsive reporter gene when fused to the LexA DNA binding domain. In addition, CAF1 contains several structural features commonly found in transcription factors, e.g., a proline-rich region, several glutamnine-rich regions, and a serine/threonine-rich region. (Sakai, A. et al. (1992) Nuc. Acids Res. 20: 6227-6233; Draper, M. P. et al. (1995) supra.)
A second CCR4 associated factor, CAF2, is a yeast protein kinase that was first identified as DBF2 and shown to be required for cell cycle progression. Immunoprecipitation and yeast two hybrid studies demonstrated that CCR4, CAF1, and CAF2 associate in vivo to form a stable complex. In addition, mutations in the genes encoding CCR4, CAF1, or CAF2 result in a similar set of pleiotropic phenotypes, including specific transcriptional defects and cell cycle progression defects. For example, mutations in any of the three genes can suppress the elevated expression of the ADH2 and HIS4-912 genes that occurs in spt6 and spt10 mutants. Taken together, the results suggest that CAF1, CAF2, and CCR4 function together as components of an evolutionarily conserved multi-protein complex that regulates transcription of numerous genes. (Draper, M. P. et al. (1995) supra.; Liu, H. Y., et al. (1997) EMBO J. 16(17):5289-5298.)
Defects in transcriptional regulation are known to contribute to oncogenesis, presumably through their affects on the expression of genes involved in cell proliferation. For example, mutant forms of transcription factors encoded by proto-oncogenes, e.g., Fos, Jun, Myc, Re1, and Spi1, may be oncogenic due to increased stimulation of cell proliferation. Conversely, mutant forms of transcription factors encoded by tumor suppressor genes, e.g., p53, RB1, and WT1, may be oncogenic due to decreased inhibition of cell proliferation. (Latchman, D. (1995) Gene Regulation: A Eukaryotic Perspective, 2nd ed. Chapman and Hall, London, UK, pp 242-255.)
The discovery of a new CAF1-related protein and the polynucleotides encoding it satisfies a need in the art by providing new compositions which are useful in the diagnosis, treatment, and prevention disorders associated with cell proliferation and inflammation.
The present invention is based upon the discovery of a new CAF1 related protein, herein after referred to as xe2x80x9cCAFRPxe2x80x9d. The invention features a substantially purified polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention further provides a substantially purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
Additionally, the invention provides a composition comprising a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention further provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purified polynucleotide which is complementary to the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides an isolated and purified polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2, and an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2. The invention also provides an isolated and purified polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2 or a fragment of SEQ ID NO:2.
The invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect, the expression vector is contained within a host cell.
The invention further provides a polynucleotide fragment useful for designing oligonucleotides or for use as a hybridization probe comprising nucleotides 1083 through 1113of SEQ ID NO:1.
The invention also provides a method for producing a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, the method comprising the steps of: (a) culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, as well as a purified agonist and a purified antagonist of the polypeptide.
The invention also provides a method for treating or preventing a disorder of cell proliferation, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for treating or preventing inflammation, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of the polypeptide having the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.
The invention also provides a method for detecting a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in a biological sample containing nucleic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to at least one of the nucleic acids of the biological sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a polynucleotide encoding the polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in the biological sample. In one aspect, the nucleic acids of the biological sample are amplified by the polymerase chain reaction prior to the hybridizing step.